Cattle–compost–soil: The transfer of antibiotic resistance in livestock agriculture

Abstract Antibiotic resistance is a major global health threat. Agricultural use of antibiotics is considered to be a main contributor to the issue, influencing both animals and humans as defined by the One Health approach. The purpose of the present study was to determine the abundance of antibiotic‐resistant bacterial populations and the overall bacterial diversity of cattle farm soils that have been treated with animal manure compost. Soil and manure samples were collected from different sites at Tullimba farm, NSW. Cultures were grown from these samples in the presence of 11 commonly used antibiotics and antibiotic‐resistant bacteria (ARB) colonies were identified. Soil and manure bacterial diversity was also determined using 16S ribosomal RNA next‐generation sequencing. Results showed that ARB abundance was greatest in fresh manure and significantly lower in composted manure. However, the application of composted manure on paddock soil led to a significant increase in soil ARB abundance. Of the antibiotics tested, the number of ARB in each sample was greatest for antibiotics that inhibited the bacterial cell wall and protein synthesis. Collectively, these results suggest that the transfer of antibiotic resistance from composted animal manure to soil may not be solely mediated through the application of live bacteria and highlight the need for further research into the mechanism of antibiotic resistance transfer.

Growth-promoting antibiotics are given at subtherapeutic doses.
They improve feed conversion and animal growth and reduced morbidity and mortality due to clinical and subclinical diseases. The average growth improvement was estimated to be between 4% and 8%, and feed utilization was improved by 2%-5% (Butaye et al., 2003).
One commonly used class of growth promoter antibiotics is ionophores. Ionophores are a diverse class of antibiotics, largely produced by the bacterial genus Streptomyces (Badger et al., 2020;Butaye et al., 2003;Wong, 2019). They are deemed as not medically important as they are not used in human medicine. In the United States, 4.6 million kilograms of ionophores were sold in 2016 with this number expected to increase significantly (Wong, 2019). In Australia, cattle farmers reported the ionophore monensin as the most prevalently used in-feed growth promoter (Badger et al., 2020).
Although the underpinning mechanisms are still unknown, ionophores assume to contribute to growth promotion through alterations to ruminal fermentation (Badger et al., 2020;Butaye et al., 2003), presumably due to changes in the gut microbiome.
Ionophores may also have direct impacts on the host's metabolism and physiology (Armstrong & Spears, 1988).
The consequence of prolonged antibiotic use in the livestock industry is the widespread increase of antibiotic-resistant populations of gastrointestinal tract microbial communities in production animals.
If livestock are prophylactically treated with the same antibiotics used to treat bacterial infections in human medicine, then a reservoir for the ARB and ARG increase within the human food supply chain (CSIRO, 2023;Sazykin et al., 2021;Tian et al., 2021). Moreover, even the use of antibiotics that are not used in humans, like ionophores, still carry risk, due to the possibility of cross-resistance or coselection (Butaye et al., 2003;Wong, 2019), for example, resistance to monensin was found to be correlated with resistance to the antibiotic vancomycin. Not only does this pose a threat to humans directly consuming animal products, but ARB and ARGs that are released in animal feces are also capable of spreading through the environment (CSIRO, 2023;Lekshmi et al., 2017). It is estimated that up to 95% of the antibiotics fed to animals are excreted in animal manure (Gutiérrez et al., 2010) and that a significant fraction of these excreted compounds maintains their activity within the soil (Gaballah et al., 2021). From a microbial ecology point of view, these excreted antibiotics act as a selective pressure in the microbial community of the soil, reduce the overall microbial diversity of the soil, and enhance the abundance of ARB in the soil. From the soil, antibiotic resistance can spread further into crops intended for human consumption and/ or to water reservoirs (Gaballah et al., 2021). This phenomenon is best described by the One Health approach, a conceptual framework that refers to the relationship between the health of humans, animals, and the environment (One Health Commission 2021; McEwen & Collignon, 2018).
In some farming operations, animal manure is stored and composted. During composting, the manure undergoes physical and nutritional profile changes. Hence, the composting process may also affect the microbial populations in the manure and has been shown to reduce the abundance of human pathogens and antibioticresistant microorganisms (Gaballah et al., 2021;Pu et al., 2019;J. Wang et al., 2015;L. Wang et al., 2019). Agricultural soils are treated with animal manure compost at high frequency and thus represent a significant ecological reservoir for ARB and ARGs, containing more than 30% of the known ARGs in public databases (Nesme & Simonet, 2015). Therefore, it is hypothesized that the soil that has been treated with composted manure from feedlot cattlefed monensin-supplemented rations will have lower microbial diversity as well as an increased abundance of ARB compared to uncomposted soil. To this end, bacterial diversity and the prevalence of ARB populations were evaluated in cattle farm soil that was treated with compost compared to control soils. The antibiotic resistance survey included 11 different antibiotics commonly used in agriculture and/or human health. Manure samples were collected from the different sites. Sixty subsamples of fresh manure (i.e., still soft and warm) were collected from the six sites (paddock and feedlot), each site grazing 50-100 head of cattle. Thirty subsamples of fresh manure from paddock sites were collected and 10 subsamples were combined into a composite sample for testing. Similarly, 30 subsamples of fresh manure from feedlot were also collected and 10 subsamples were combined into a single sample for testing. While 100 manure subsamples were T A B L E 1 Standard diet formulation feed to cattle on the Tullimba feedlot/farm.

| Isolation of ARB from field samples
Suspensions of soil and manure samples were made by placing 5 g of soil or manure into a sterile flask with 45 mL of sterile distilled water, shaken at 28°C, 220 rpm for 1 h  and the slurries were filtered through sterile gauze. The resuspended soil and compost filtrates were serially diluted from 10 −1 to 10 −4 , in sterile distilled water under aseptic conditions.

| Molecular identification of bacterial species isolated from soil and manure
The genomic DNA of ARB isolates was extracted using the Isolate II Genomic DNA Kit (Bioline), according to the manufacturer's instructions. The quantity and concentration of DNA were determined using a Nanodrop 8000c spectrophotometer (Thermofisher Scientific). The bacterial 16S ribosomal RNA (rRNA) gene was amplified using primer pairs (1369-F, 5′-CGGTGAATACGTTCYCGG-3′ and 1541-R, 5′-AAG GAGGTGATCCRGCCGCA-3′) as described previously (Huang et al., 2015). The PCR reaction and amplification program were described in Anisimova & Yarullina (2019). The PCR products were run on a 1% agarose gel and stained with GelRed (Biotium). The PCR product was purified using a QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions and was sequenced at Australian Genome Research Facility using Sanger sequencing technology. Basic Local Alignments Tool (BLAST) algorithm was used to analyze sequence data.

| Analysis of bacterial diversity
Metabarcode data were imported to R Core Team (2021;  Alpha-and beta-diversity analysis of metabarcoding data was performed on the relative abundance of 16S ASVs after samples were rarefied to a common depth (3155 reads per sample).
Shannon-Wiener indices were calculated using the vegan package (Oksanen et al., 2020), and the difference between treatments was assessed by the Kruskal-Wallis rank-sum test, followed by pairwise comparison with Dunn's test. Bray-Curtis distances were calculated with vegan (Oksanen et al., 2020), and visualized with ape (Paradis & Schliep, 2019) and differences between treatments were assessed through permutational multivariate ANOVA (999 permutations).

| Plasmid extraction and bacterial transformation
The plasmid pUCITD-AMP Golden Gate plasmid containing the synthetic erythromycin resistance gene (ermF)   significantly greater compared to control, untreated soil but the bacterial diversity structure of the compost-treated and control bacterial soil communities was different.

| 16S identification of resistant isolates
The 16S sequence analysis was used to identify the ARB isolates from compost-treated and control soils as well as from fresh manure Bacillus (NR_152692.1) isolates were performed. Figure 4 illustrated that a plasmid was successfully extracted from Bacillus (NR_152692.1). When this plasmid was transformed to antibioticsusceptible DH5α E. coli, it conferred antibiotic resistance activity against erythromycin but not to gentamycin, tetracycline, or streptomycin ( Figure 4 and  animal manure revealed a decreasing abundance of ARB from feedlot to paddock and finally stored manure, which contained low ARB abundance. In agreement with a previous study, animal manure, particularly cattle manure, contains a substantial number of ARB even if the animal has not received antibiotics previously (Pu et al., 2019;Udikovic-Kolic et al., 2014). Additionally, higher ARB loads have also been attributed to cattle in feedlots due to the accumulation of antibiotic residue in animal products (Chee-Sanford et al., 2009;Heuer et al., 2011) as well as the limited space leading to increased transmission of ARB via direct contact among animals or through the ingestion of fecal contamination in feed and water sources (Netthisinghe et al., 2013). These conditions may act to apply selection pressure, thereby reducing the overall bacterial diversity of feedlot cattle compared to fresh manure from cattle grazed on paddocks, as has been observed here and in previous studies (Chang et al., 2021;Gaballah et al., 2021;Khan et al., 2008).
However, composting manure significantly decreased the abundance and diversity of ARB mainly due to altered environmental conditions such as sunlight, temperature, pH values, and moisture, which all play a significant role in the degradation of antibiotics and the survival of the microorganisms (Chee-Sanford et al., 2009;Koike et al., 2007;Martinez, 2009;Netthisinghe et al., 2013;Sengeløv, 2003). For these reasons, this study observed that the antibiotic resistance rates of bacteria in stored manure were very low compared to fresh manure from feedlot sites.
Spreading composted manure on paddocks produced divergent bacterial communities with a significantly higher abundance of ARB.
This is in accordance with other observations in the literature (Pu et al., 2019;L. Wang et al., 2019). Considering that the compost treatment contained low amounts of live ARB, this raises the question, what is the resistance transfer mechanism, if it is not through the application of live ARB?
One hypothesis is that antibiotic resistance is transferred via resistant DNA or ARG mobile elements. Previous studies demonstrated that ARG and mobile genetic elements remain in the compost material following aerobic composting, long after the bacterial decay (Pu et al., 2019;Xie et al., 2016;Zhang et al., 2016). Once spread on the soil, ARG may disseminate and reach the soil microbial community, conferring antibiotic resistance through horizontal gene transfer (Pu et al., 2019). Analysis of the common resistant isolates identified in this study revealed a novel plasmid carrying the erythromycin resistance gene, with demonstrated capacity to confer resistance to erythromycin in other microbes from different genera.
Further analysis should be aimed at characterizing antibiotic resistance-carrying mobile genetic elements that are present in compost.
Another mechanism could be due to antibiotic residues within the animal manure that are introduced into the soil with compost T A B L E 2 Zones of inhibition for each antibiotic that was tested in the disk diffusion assay when confirming resistance to erythromycin in the transformed DH5α Escherichia coli cells.  Xie et al., 2016). Further studies are needed to understand the mechanisms of antibiotic resistance transfer from compost to the soil and to develop strategies to minimize this process. Interestingly, compost application not only acted to increase ARB abundance but also influenced the overall diversity of the soil microbial population. Further research is needed to identify which components in the compost are the main facilitators of this effect.

| Effect of compost treatment on the development of specific antibiotic resistance
The highest number of ARB in manure samples from feedlot and compost samples was for monensin (Figure 2). This is not surprising as monensin was given prophylactically to the cattle. Following monensin, high resistance was observed for the antibiotics, cefotaxime, and ampicillin, which inhibit cell wall synthesis ( Figure 2). This is consistent with previous investigations that reported how bacterial isolates from cattle manure such as Salmonella spp., E. coli, and Pseudomonas spp. showed high resistance levels for ampicillin, cefotaxime, and monensin (Camiade et al., 2020;Ibrahim et al., 2016;Xu et al., 2018).
Interestingly, we detected a high abundance of ARBs in the untreated soil. In the case of tetracycline, ARB abundance in the untreated soil was higher than the one observed in the treated soil ( Figure 3). This high resistance may be a result of historical antibiotic use, before the documented history of these soils; alternatively, this soil could be naturally high in tetracycline-resistant bacteria whose abundance was decreased in the treated soils as a result of continuing compost application.

| CONCLUSION
In conclusion, the current study demonstrates that the application of cattle manure compost as a soil treatment acts to increase the abundance of antimicrobial resistance in soil and reduce the microbial diversity of the soil. This contributes to the global increase in antibiotic resistance, which has extended implications on both animal and human health, as defined by the one health framework. This study highlights the importance of understanding the transfer mechanism for antibiotic resistance from manure to the soil to maintain soil fertilization without enhancing antibiotic resistance.

ACKNOWLEDGMENTS
The authors